The identity of the cognate lipid ligands for the PLUNC (palate, lung, and nasal epithelium clone) protein family have remained elusive since the family was discovered more than one decade ago. The most characterized member of the family, the short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is abundantly expressed by normal airway epithelial cells. Due to its distribution in allergic airways and sequence similarity to BPI (bactericidal/permeability increasing protein), its function in the innae immune system has become of great interest. SPLUNC1 plays crucial roles in host defense against pathogen infections. SPLUNC1 has bactericidal activity against Mycoplasma pneumoniae and arrests the growth of Pseudomonas aeruginosa; the n-terminal domain inhibits the epithelial sodium channel (ENaC), and it is also reported that SPLUNC1 acts as surfactant to reduce airway surface tension and interfere with biofilm formation by pathogens. However, no definitive evidence has shown that SPLUNC1 functions similarly to BPI, including no apparent bactericidal activity against most Gram-negative bacteria or Gram-positive bacteria, no neutralization of LPS, and no opsonin activity. Furthermore, there are controversial reports about the binding between SPLUNC1 and LPS. To better understand the function roles of SPLUNC1 in host defense and innate immune system, we recently determined the high-resolution structure of SPLUNC1. To our surprise, there are dramatic structural differences between BPI and SPLUNC1. The overall structure is similar to that of Der p 7, a house-dust mite allergen. In particular, the surface of SPLUNC1 is covered with negatively charged patches, in contrast to the positively charged surface of BPI, which is essential to bind the negatively charged head group of LPS. Our in vitro binding assays showed no binding between SPLUNC1 and LPS. To identify potential lipids that might bind to SPLUNC1, we subjected SPLUNC1 derived from human 293 cells to mass spectrometry analysis and found that SPLUNC1 was saturated mainly with Sphingomyelin(SM) and minorly with Phosphatidyl choline (PC). In vitro binding assays proved that SPLUNC1 could bind Sphingomyelin but not the most common PC (POPC from chicken egg). Lipid binding screen revealed that DPPC, the most rich lipid surfactant in lung, binds to SPLUNC1. Furthermore, DPPC is the only lipid of lipid extracts from human and mouse BAL fluid that binds SPLUNC1 while SM is undetectable. Interestingly, SPLUNC1 is the first identified protein receptor of DPPC, which is the only active surface component of lung surfactant capable of lowering surface tension to near zero. We hypothesize that DPPC could be the cognate lipid ligand for SPLUNC1 but not LPS. Thus, the first goal of this proposal is to identify, verify, and confirm the cognate lipid ligands of SPLUNC. On the other hand, information regarding the specific binding between proteins and lipids is also limited. We have carried out systemic screening of potential targets for SPLUNC1. Our preliminary data showed that the specificity between ligand and protein is not only determined by the head group of the lipid but also the by the fatty acid chains. Accordingly, the second goal of this proposal i to elucidate the structural basis of the specificity determinants between SPLUNC1 and lipid(s). Based on structural features of the lipid identified and the general specific interaction determinants of SPLUNC1 with lipids, we will proceed with investigating potential lipid ligands for other PLUNC family members. Antimicrobial peptides (AMP) are key weapons by which eukaryotes protect themselves against infection and represent a major component of the innate immune system. This proposal will investigate the SPLUNC1 protein, a member of the poorly characterized PLUNC family of AMPs. SPLUNC1 is dramatically down regulated in patients with Asthma, COPD, and Cystic Fibrosis diseases. DPPC is highly elevated in Asthma. Impaired sphingolipid synthesis causes airway hyperreactivity while SM is a major product of sphingolipid metabolism process. Determining the novel mechanisms of SPLUNC1 regulation and function in the airways will improve our understanding of impaired innate immunity in allergic airways. By translating our research discoveries into therapies through manipulation of SPLUNC1, DPPC, and SM, our proposed work will ultimately provide opportunities to treat bacterial or other pathogen infections in chronic lung diseases such as asthma and other pulmonary diseases. Our discovery of DPPC as the cognate receptor of SPLUNC1 while ruling out LPS will also bring great impact in the PLUNC field, which may completely change the direction of the research focus of the field.